A Safety Protocol written for a biotech company  

          Company and Scientists names have been removed for privacy and non-disclosure purposes

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SAFETY PROTOCOL

 Protocol for Handling RBCs Containing Inactivated HIV

The following protocol addresses the biotech company's in-house handling of the inactivated HIV-loaded samples that will be generated from the Collaborating Laboratory Study.  The samples will consist of cytofixed mouse RBCs that have been exposed to inactivated HIV, in the presence of HIV-Confidential Drug, HIV-mAb or PBS. A total of 54 samples are expected.

     Samples will be collected and processed (cytofixed) at Collaborating Laboratory prior to transport to laboratory.

Sample Processing at cooperating laboratory

        Blood samples from inactivated HIV injected mice will be received from Dr. ________

        The RBCs will be centrifuged and 1ml of plasma will be transferred for shipment to diagnostic laboratories.

        200ul of plasma will be stored frozen at -80⁰C.

        RBC pellets will be resuspended in 1mL PBS and 50uL transferred to a fresh tube.

        These samples will be re-spun and then resuspended in 1mL Cytofix Buffer at a 1:4

        dilution.

        The samples will be cytofixed for 30mins at 4⁰C.

        After 30mins the samples will be washed and resuspended in 400uL PBS/BSA 1% and

        Transported to biotech company on ice for staining and FACScalibur analysis.

Preparation of Samples (Cytofixing) Performed at Cooperating Lab before transport to biotech company.

Handling of Samples at Biotech Company

1. Samples will be transported to biotech company Virology in a sealed, cooled container.
2. Samples will be admitted to Virology through the rear entrance, ensuring that they will only traverse laboratory space.
3. Samples will be carried directly into Virology and immediately placed in the BioSafety Hood, or stored in the Virology Refrigerator. (The Lab will once again be key-card access only)
4. Staining of samples will be carried out within the BioSafety Hood, to include shaking incubations.
5. When necessary, samples will be tightly capped and moved to the table-top centrifuge for the wash steps, and then immediately returned to the hood for any further processing.
6. When staining is complete, samples will be tightly capped and covered and carried to the FACScalibur.
7. Following the FACScalibur analysis of samples, the FACScalibur will be allowed to go through its normal cleaning cycle.
8. Following this cleaning cycle, a second manual bleach cycle will be run for 20mins, followed by 10min. water flush.
9. The samples will be immediately capped when the FACS run is complete, returned to Virology, and immediately decontaminated in 10% bleach, in the Hood, overnight.
10. Following decontamination, the sample tubes will be tightly capped and discarded in a sealed mini-sharps container.
11. The Virology Laboratory, equipment, biosafety cabinet, centrifuge, shaker and all surfaces will be decontaminated with a 10% bleach solution, following which all surfaces will be wiped down with 50% ETOH.